Composition for gene transfer into cells

ABSTRACT

The present invention is directed to a composition for gene transfer which composition contains a quaternary ammonium salt represented by formula (1):  
                 
 
     (wherein each of R 1 , R 2 , R 3 , R 4 , and R 5 , which are identical to or different from one another, represents a C9-C17 aliphatic group); X 1  represents a halogen atom; and n is an integer from 1 to 10 inclusive; and a method for introducing a gene into a cell by use of the composition.  
     The composition enables effective delivery and expression of a gene which previously could not be effectively expressed in cells due to the low ratio at which the gene is delivered into cells. Therefore, the composition is advantageously used as a gene transfer reagent or a pharmaceutical.

TECHNICAL FIELD

[0001] The present invention relates to a composition for gene transferinto cells, as well as to a method for gene transfer into cells by useof the composition.

BACKGROUND ART

[0002] Plasma membranes have low permeability to some compounds whichare used as drugs, and thus the drugs fail to exhibit sufficientintracellular pharmacological effect. Low plasma membrane permeabilitymay be attributable to, for example, the compound having low lipidsolubility or high molecular weight. A typical example of such acompound to which plasma membranes exhibit low permeability is a gene.

[0003] At present, therapeutic treatment by use of such drugs to whichplasma membranes exhibit low permeability, in particular, a gene, isconducted by way of injection, etc. However, the permeation of such adrug into the inside of cells is so poor that it is unable to providesatisfactory therapeutic effect.

[0004] To solve this problem, there have been proposed variousconventional techniques known as drug delivery systems (DDS). As suchsystems there are, for example, liposomes formed primarily ofphospholipids, emulsions composed of surfactants and oils such assoybean oil, mixed micelles made of lipids and surfactants, andmicrocapsules/microspheres made of biodegradable or non-degradablepolymers. However, the conventional techniques have failed to increasepermeability of a drug through the membrane; rather, in vitro evaluationhas revealed that conventional drug delivery systems have an effect ofdecreasing cell-membrane permeability of a drug. This is because thedrug itself is encapsulated in the drug delivery system, and release ofthe drug from the system serves as a determining factor. Despite thisdrawback, drug delivery systems have been attracting close attention,and have been applied to many drugs. This is because when a drug isencapsulated in a drug delivery system, in vivo degradation of the drugcan be suppressed, and in vivo kinetics of the drug can be controlled,thus eventually increasing the in vivo drug concentration in thevicinity of the target tissue or cells. Even in the case of liposomes,one of the typical drug delivery systems, although suppression of drugdegradation and controlling the in vivo kinetics can both be achievedwith relative ease, particles of liposomes ultimately accumulate in thevicinity of the target tissue or cells at high concentration and releasethe drug, and the remainder of the drug delivery steps depend solely onthe permeability of the drug through the plasma membrane. Thus, althoughliposomes can attain an increased drug concentration near target cells,they have no effect on the permeability of the drug through the plasmamembrane.

[0005] In some in vitro cases, a drug delivery system increases the rateof drug delivery into cells. Example cases include use of phagocyticcells such as macrophages and monocytes. Phagocytic cells readily ingestmicroparticles, such as liposomes, by endocytosis, and therefore,transferability of the drug into cells may be increased if the drug isencapsulated in a drug delivery system rather than administered alone.In this case, permeability of the drug through the plasma membrane isnot increased. However, if the drug can be temporarily incorporated intocellular vesicles, such as endosomes and lysosomes, together with thedrug delivery system and happens to be stable in thesemicroenvironments, the drug can further enter the cytoplasm, resultingin increased drug transferability into cells.

[0006] Also, in recent years, extensive research has been directed togene transfer into non-phagocytic cells, which is achieved throughformation of a complex with a gene and cationic lipids (or liposomescontaining the cationic lipids) or through encapsulation of a gene intoliposomes containing the cationic lipids, thereby allowing the gene tobe expressed within the cells. Even though almost nothing is known aboutthe gene transfer mechanism into cells, reagents related to theabove-described research are widely commercialized (including reagentssuch as lipofectAMINE, lipofectACE, lipofectin, transfectam, andgenetransfer). Presently, biological researchers are using thesereagents on a daily basis as very useful tools for gene transfer intocells, and this method serves as a substitute for the virus andmicroinjection methods. However, these commercialized reagents have manydrawbacks as described in the following a) to d). a) These reagents, ascommercialized products, are not stable, and thus are not suitable forstorage. Many of these commercialized products are sold in the form of adispersion in water, and the pH of their aqueous solvents are usuallyvery low pH (for example, the pH is 3.5 for lipofectAMINE andlipofectACE, and 4.3 for lipofectin). Because of this low pH, lipidstend to degrade during storage. It has often been pointed out thatefficiencies of gene transfer into cells and of gene expression by useof liposomes, etc. do not have satisfactory reproducibility. One reasonfor this is the inherent instability of the products. (b) Anotherdrawback is that those products are very unstable in the presence offetal bovine serum added to a medium for cell culture. As a matter offact, the commercialized products employ the following protocol for genetransfer: Before gene transfer, the cultured medium containing fetalbovine serum is replaced with serum-free medium; and then, aftercompletion of the gene transfer, the serum-free medium is replaced byserum-containing medium. Recently, it has become clear that thesecommercialized products are also very unstable in blood as well as invivo. (c) A further drawback is that those commercialized products arenot suitably designed for easy handling. Many of the commercializedproducts, such as lipofectAMINE, lipofectACE, and lipofectin, areprovided in the form of a dispersion in water. For gene delivery,aqueous solvents that contain gene samples are added to these products.However, this protocol only allows the products to form complexes withthe genes where genes bind only to the outside of the liposomes.Therefore, these products cannot yield vesicles having the genesencapsulated inside the liposomes. (d) Moreover, a further drawback isvery strong cytotoxicity originated from those products. As is wellknown, the primary purpose for which biological researchers use thesecommercialized gene transfer reagents is to obtain the cells that havebeen transformed with exogenous genes and are capable of expressingthose genes, and to subsequently use the obtained cells in subsequentstudies. For such a purpose, in most cases, whether or not a minorportion of cell population dies during the gene transfer step isimmaterial, so long as such transformed cells can be obtained. Thus,there have been commercialized some reagents making use of cationiclipids alone or in combination with liposomes, for delivering into cellsa drug such as gene which in nature cannot be permeated through themembrane. However, those commercialized reagents involve many problems,as mentioned above. Thus, it is no exaggeration to say that applicationof such reagents to human use (such as gene therapy) is unthinkable atpresent. (Note: Gene therapy includes ex vivo and in vivo methods. Inthe case of ex vivo treatment, in which cells are taken out of apatient, treated in vitro, and subsequently returned to the patient,cytotoxicity raises a great problem.)

[0007] As pointed out above, it would be no exaggeration to concludethat there is no conventional satisfactory method whereby a gene—which,except for special cases such as the case of phagocytic cells or somecommercially available gene transfer reagents, is poorly permeatedthrough the plasma membrane, is poorly delivered into the inside ofcells, or encounters difficulty in manifesting its activity inside thecells—can be delivered into cells, after which the gene is allowed toexert its pharmacological efficacy.

[0008] Thus, an object of the present invention is to improve permeationthrough a plasma membrane, transmembrane delivery, and intracellularexpression of a gene, to which the plasma membrane has low permeability,in order to deliver the gene into the inside of cells, or to express thegene within the cells.

DISCLOSURE OF THE INVENTION

[0009] In view of the above, the present inventors have performeddiligent studies in order to solve the problems involved in delivery ofa gene to the inside of cells whose plasma membranes have lowpermeability to the gene, and further to improve expression of the geneinside the cell. As a result, the inventors have found thatadministration of a gene together with a quaternary ammonium saltrepresented by formula (1) to cells leads to efficient gene expression,not only in vitro but also in vivo. The present invention has beenachieved on the basis of this finding.

[0010] Accordingly, the present invention provides a composition forgene transfer into cells, which composition comprises a quaternaryammonium salt represented by the following formula (1):

[0011] (wherein each of R¹, R², R³, R⁴, and R⁵, which are identical toor different from one another, represents a C9-C17 aliphatic group); X¹represents a halogen atom; and n is an integer from 1 to 10 inclusive.

[0012] The present invention also provides a composition for genetransfer into cells, which composition comprises a quaternary ammoniumsalt represented by formula (1) and a gene.

[0013] The present invention also provides a gene transfer methodcomprising applying a composition containing a quaternary ammonium saltrepresented by formula (1) and a gene into a cell either in vivo or invitro.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] In formula (1), which represents the quaternary ammonium salt tobe incorporated into the composition of the present invention, examplesof C9-C17 aliphatic groups represented by R¹, R², R³, R⁴, and R⁵ includelinear or branched, saturated or unsaturated C9-C17 aliphatic groups.Among them, linear or branched C9-C17 alkyl groups are preferred, andlinear or branched C11-C15 alkyl groups are more preferred. Also, C9-C17linear alkyl groups are more preferred, with C11-C15 linear alkyl groupsbeing particularly preferred. Specifically, undecyl, tridecyl, andpentadecyl linear alkyl groups are particularly preferred. R¹, R², R³,R⁴, and R⁵ may be identical to or different from one another. However,identical groups are preferred from the point of view of manufacturing.

[0015] In formula (1), the halogen atom represented by X¹ is notparticularly limited. However, chlorine or bromine is preferred.

[0016] In formula (1), n represents an integer from 1 to 10 inclusive.Among such integers, 1 and 10 are particularly preferred. When n is 1, Ais preferably

[0017] Also, when n is 10, A is

[0018] In the composition for gene transfer according to the presentinvention, the amount of the quaternary ammonium salt represented byformula (1) varies in accordance with the gene employed, use of thecomposition, and the physical form of the composition. Basically, anyamount that allows the gene to be transferred into cells is sufficient.For example, the weight ratio of the composition to the gene ispreferably 1:1 to 1:1000; more preferably 1:1-1:100.

[0019] Genes used in the present invention may be in the form of eitheroligonucleotides, DNA, or RNA. In particular, genes resulting intransformation upon in vitro gene transfer and those becoming activeupon in viva gene expression are preferred. Examples for the latter caseof in viva expression include those for gene therapy and breeding ofindustrial animals, such as domestic animals and animals forexperimental use. When genes used for gene therapy are incorporated intothe composition of the present invention, the composition serves as apharmaceutical composition. Examples of genes for such gene therapyinclude antisense oligonucleotides, antisense DNA, antisense RNA, andgenes encoding physiologically active proteins such as enzymes andcytokines. When genes encoding a certain enzyme are used, a substancethat exhibits pharmacological effect due to the action of the enzyme maybe used in combination with such genes. For example, tumors may betreated by first delivering a thymidine kinase gene in advance andcausing expression in vivo (in tumors), and subsequently administeredacyclovir can kill the tumors.

[0020] In order to improve the efficiency of gene transfer, thecomposition of the present invention may further contain phospholipidsand/or cholesterol. Examples of phospholipids which may be containedinclude phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,cardiolipin, sphingomyelin, plasmalogen, and phosphatidic acid. Thesephospholipids may be used singly or in combination. Preferably,phosphatidylethanolamine and phosphatidylcholine are used singly or incombination of two or more species; and use of phosphatidylethanolamineis particularly preferred. Fatty acid residues of these phospholipidsare not particularly limited. Preferable fatty acid residues includeC12-C18 saturated or unsaturated fatty acid residues; and the palmitoylgroup, oleoyl group, stearoyl group, and linoleyl group are mostpreferred. The amount of the phospholipid to be incorporated into thecomposition of the present invention is preferably 0-80%, morepreferably 10-70%, particularly preferably 25-70% based on the molefraction. In the case of cholesterol, the amount is preferably 0-70%,more preferably 10-60%, particularly preferably 20-50%, based on themole fraction.

[0021] When the quaternary ammonium salt represented by formula (1) isused together with the phospholipid and/or cholesterol, the efficiencyof gene transfer increases significantly as compared with the case ofsole use of the quaternary ammonium salt. Particularly, a remarkableincrease in efficiency is observed when the phospholipid and thequaternary ammonium salt represented by formula (1) are used incombination. Also, in formula (1), when A is a quaternary ammonium saltrepresented by

[0022] use in combination with phospholipid provides a remarkableincrease in the efficiency of gene transfer.

[0023] The amount of the quaternary ammonium salt to be incorporatedinto the composition of the present invention is, based on the molefraction, preferably 5-100%, more preferably 10-75%, particularlypreferably 15-50%.

[0024] When the composition of the present invention contains thequaternary ammonium salt, and phospholipid or cholesterol, the moleratio of the quaternary ammonium salt to the phospholipid or cholesterolis preferably 1:9-9:1, more preferably 2:8-8:2, particularly preferably3:7-7:3. In this case, one type of phospholipid or a mixture of two ormore types of phospholipid can be used.

[0025] When the composition of the present invention contains thequaternary ammonium salt, phospholipid, and cholesterol, the mole ratioof the mixture of the quaternary ammonium salt and phospholipid to thecholesterol is preferably 3:7-9:1, more preferably 4:6-9:1, particularlypreferably 5:5-8:2. In this case, one type of phospholipid or a mixtureof two or more types of phospholipid can be used.

[0026] Further, lipid-soluble vitamins such as vitamin E can beincorporated into the composition of the present invention.

[0027] With regard to the mode of the composition of the presentinvention, the quaternary ammonium salt (1) can be incorporated alone orcan simply be mixed with the phospholipid and/or cholesterol. Also, inorder to form a phospholipid membrane structure, the quaternary ammoniumsalt (1) can be incorporated alone or can be incorporated in combinationwith the phospholipid and/or cholesterol. Although no particularlimitation is imposed on the modes and manufacturing methods for thelipid membrane structure, examples thereof include a dried lipidmixture, a lipid mixture dispersed in an aqueous solvent, a lipidmixture dispersed in an aqueous solvent and further dried, and a lipidmixture dispersed in an aqueous solvent and then frozen.

[0028] Concerning the method of manufacturing the dried lipid mixture,for example, lipid components to be used for the mixture are firstdissolved with an organic solvent such as chloroform, and subsequentlysubjected to in vacuo drying by use of an evaporator or to spray-dryingby use of a spray-dryer.

[0029] With regard to the dispersion mode in an aqueous solvent, thoughno particular limitation is imposed on the lipid membrane structure,examples thereof include multilamellar liposome, unilamellar liposome,O/W emulsion, W/O/W emulsion, spherical micelle, and string-shapedmicelle, as well as an amorphous multi-layered structure. Although noparticular limitation is imposed on the particle size of the lipidmembrane structure, the diameter of the liposomes and emulsions is 50 nmto several μm, and that of the spherical micelle is 5 nm to 50 nm.However, in the case in which the concept of a diameter cannot beapplied to the string-shaped micelle and amorphous multilayer structure,the concept of the thickness of one layer can be employed, which is 5 nmto 10 nm, and layers are stacked one on another, thus forming theamorphous multilayer structure.

[0030] No particular limitation is imposed on the aqueous solvent.However, in addition to water, examples thereof include the following:sugar solutions such as those containing glucose, lactose, or sucrose;polyalcohol solutions such as those containing glycerin or propyleneglycol; physiological saline; buffered solutions such asphosphate-buffered solutions, citrate-buffered solutions, andphosphate-buffered physiological saline; and media for cell culture. Inorder to effect long-term stable preservation of lipid membranestructure in the dispersion mode in an aqueous solvent, the followingpoints are important: from the physical viewpoint, such as prevention ofaggregation, electrolytes are eliminated from the aqueous solvent to thegreatest possible extent; and from the viewpoint of lipid chemicalstability, the pH of the aqueous solvent is set to a range from weakacidity to neutral (pH3.0-8.0) and dissolved oxygen is removed from thesolvent by means of bubbling with nitrogen. Further, use of sugarsolutions for preservation of lyophilized samples and spray-driedsamples, as well as use of sugar solutions and polyalcohol solutions forcryopreservation, can achieve effective preservation.

[0031] Although no particular limitation is imposed on theconcentrations of such aqueous solvents, sugar solutions preferably haveconcentrations of 2-20% (W/V) more preferably 5-10% (W/V); polyalcoholsolutions preferably have concentrations of 1-5% (W/V), more preferably2-2.5% (W/V); and buffered solutions preferably have concentrations of5-50 mM, more preferably 10-20 mM.

[0032] Also, no particular limitation is imposed on concentrations ofthe lipids forming the lipid membrane structure in the aqueous solvent.The total lipid concentration of the quaternary ammonium salt (1),phospholipid, and cholesterol used for the lipid membrane structure ispreferably 0.001 mM-100 mM, more preferably 0.01 mM-20 mM.

[0033] In order to manufacture the dispersion mode of the lipid membranestructure in the aqueous solvent, an aqueous solvent is added to theabove-described dried lipid mixture, followed by emulsification by useof an ultrasonic homogenizer, a high-pressure jet homogenizer, or anemulsifier such as a homogenizer. Alternatively, without use of suchdried lipid mixtures, a well-known liposome manufacturing method such asthe reverse-phase evaporation method can be used, and no particularlimitation is imposed on such manufacturing methods. In order to controlthe particle size, extrusion can be carried out under high pressurethrough a membrane filter having a uniform pore size.

[0034] Further, examples of the methods of drying the lipid membranestructure dispersed in an aqueous solvent include typical lyophilizationand spray drying. Concerning the aqueous solvent for this purpose, asmentioned above, sugar solutions such as a sucrose or lactose watersolution are preferred. Among the merits for further drying the lipidmembrane structure already manufactured, such dried forms allowlong-term preservation of the lipid membrane structure. Additionally,when gene-containing solutions are added to the dried forms, the lipidmembrane structure is efficiently rehydrated so that the gene is alsoeffectively retained by the lipids forming the lipid membrane structuresuch as liposomes.

[0035] A conventional method may be used for further freezing thedispersion mode of the lipid membrane structure in the aqueous solvent.As described above, this method preferably employs aqueous solvents suchas a sugar solution or a polyalcohol solution. The merits of furtherfreezing the lipid membrane structure include permitting long-termpreservation of the lipid membrane structure.

[0036] The composition of the present invention which contains a gene(gene-containing composition) will next be described.

[0037] The mode of the gene-containing composition of the presentinvention may be a mixture of quaternary ammonium salt (1) and a gene; amixture of quaternary ammonium salt (1), a gene, and phospholipid and/orcholesterol; a mixture of a gene and a lipid membrane structure formedof quaternary ammonium salt (1) alone or in combination withphospholipid and/or cholesterol; or a form in which a gene is carried onthe lipid membrane structure. Here, “carry” indicates that the gene isburied in the lipid membrane, present on the surface of the lipidmembrane, present inside the membrane, buried in a lipid layer, orpresent on the lipid layer.

[0038] As is the case with the lipid membrane structure, no particularlimitation is imposed on the form and production method of thegene-containing composition. For example, the composition may be formedinto a dry mixture, a dispersion in an aqueous solvent, or a dry orfrozen form of the dispersion.

[0039] The dry mixture of a lipid and a gene may be prepared by meansof, for example, dissolving a lipid component and a gene in an organicsolvent such as chloroform, and subsequently subjecting the solution tovacuum drying by use of an evaporator or to spray-drying by use of aspray dryer.

[0040] Non-limiting examples of the dispersion of the mixture of a lipidmembrane structure and a gene in an aqueous solvent includemultilamellar liposome, unilamellar liposome, O/W emulsion, W/O/Wemulsion, spherical micelle, string-shaped micelle, or an amorphousmulti-layered structure. No particular limitation is imposed on theparticle size of the mixture, the composition of the aqueous solvent, orthe concentration of the mixture in the aqueous solvent.

[0041] Aqueous dispersions of a mixture of the lipid membrane structureand a gene may be prepared through several methods having differentcharacteristic features, yielding different forms of the resultingmixtures of the lipid membrane structure and gene.

[0042] According to the first manufacturing method, an aqueous solventis first added to the above-mentioned dry mixture of lipid and gene,followed by emulsification by use of a commonly-employed emulsifier suchas a homogenizer, ultrasonic emulsifier, or a high-pressure jethomogenizer. In order to control the particle size, extrusion may becarried out under high pressure through use of a membrane filter havinga uniform pore size. In this case, in order to prepare the dry mixtureof lipid and gene, the gene must first be dissolved in an organicsolvent. This method is advantageous in that interaction between thegene and the lipid membrane structure can be fully utilized, so that thegene can enter the inside of the multi-layer when the lipid membranestructure has a layered structure. Thus, in general, the method enablesmore genes to be carried by the lipid membrane structure.

[0043] According to the second manufacturing method, after lipidcomponents are dissolved in an organic solvent, the organic solvent isremoved so as to obtain a dry material. Then, an aqueous solvent thatcontains a gene is added to the dry material, followed byemulsification. In order to control the particle size, extrusion can becarried out under high pressure through a membrane filter having auniform pore size. This method can be applied to genes that aredifficult to dissolve in an organic solvent but are easily dissolved inan aqueous solvent. This method is advantageous in that genes can beretained by the inner aqueous phase of liposomes.

[0044] According to the third manufacturing method, an aqueous solventthat contains a gene is added to lipid membrane structures (such asliposomes, emulsions, micelles, or layered structures) which has alreadybeen dispersed in another aqueous solvent. Therefore, this method isonly applied to water-soluble genes. Further, by this method, genes arelater added separately to the lipid membrane structure that has beenprepared in advance. Because of this, if the gene is large in size, itis unable to enter the inside of the lipid membrane structure and onlybinds to the surface thereof. When liposomes are used as the lipidmembrane structure, this third method is known to provide a sandwichstructure in which the gene is sandwiched by liposomal particles(generally called a complex). The merit to this third method is that thelipid membrane structure, such as that of the liposomes, emulsions,micelles, and layered structures, dispersed in an aqueous solvent can bestored after manufacturing, and can be used not only for one type ofgene but also commonly used for other types of gene. Further, when thismethod is used, the dispersion containing only the lipid membranestructure is prepared in advance. Because of this, there is no need toconsider degradation of drug during emulsification. Also, particle sizecan be easily controlled. Thus, this manufacturing method is more easilycarried out than are the first and second methods.

[0045] According to the fourth manufacturing method, a lipid membranestructure is first dispersed in an aqueous solvent, followed by drying.An aqueous solvent that contains a gene is added to the dried material.As is the case with the third manufacturing method, this method can beapplied only to water-soluble genes. However, the third and fourthmanufacturing methods clearly differ in the state of the lipid membranestructure and gene. In the case of the fourth manufacturing method,first a lipid membrane structure dispersed in an aqueous solvent isprepared, followed by drying to obtain dried materials. After this step,the lipid membrane structure exists in a solid state as fragments of thelipid membrane. As mentioned above, obtaining such solid state lipidfragments requires use of an aqueous sugar solution, preferably asucrose or lactose solution, serving as an aqueous solvent. When anaqueous solvent that contains a gene is added to the solid state lipidfragments, they are quickly hydrated to reconstitute the lipid membranestructure, as water is absorbed. At this point, the resultingcomposition that retains the genes inside the lipid membrane structureis generated. In contrast, in the case of the third manufacturingmethod, when the gene is large in size, it is unable to enter the insideof the lipid membrane structure and only binds to its surface. Oneadvantage of the fourth manufacturing method is that once the materialsare manufactured, they can be used not only for one type of gene butalso commonly for other types of genes. Another advantage is that sincethe aqueous dispersion containing the lipid membrane structure alone isprepared in advance, degradation of the drug during emulsification isnot a consideration. Further, the particle size can be easilycontrolled. Thus, this manufacturing method is more easily carried outthan are the first and second methods. In addition, since the product isprepared by means of lyophilization or spray-drying, its storagestability is reliably ensured, enabling use as a commercial product;after rehydration of the dry product by use of a gene-containingsolution, the particle size is restored to the original size, and eventhe large gene can be easily retained inside the lipid membranestructure.

[0046] Concerning other methods for preparing the dispersion mode of themixture of the lipid membrane structure and a gene, a well-known methodfor preparation of liposomes such as the reversed-phase evaporationmethod can be used. In order to control the particle size, extrusion canbe carried out under high pressure through a membrane filter having auniform pore size. Example methods of drying the above-mentioned mixtureof the lipid membrane structure and genes dispersed in the aqueoussolvent include lyophilization and spray drying. For this aqueoussolvent, as is the case with use of the lipid membrane structure alone,a sugar solution, preferably a sucrose or lactose solution, is used.

[0047] Conventional freezing methods may be used for freezing theabove-mentioned dispersed mixture of the lipid membrane structure andgene in an aqueous solvent. This aqueous solvent is preferably a sugaror polyalcohol solution, as is the case with the lipid membranestructure alone.

[0048] The composition of the present invention can be applied not onlyto genes but also to other drugs having very low lipid solubility andreagents which are difficult to be delivered into cells, such asphysiologically active peptides of high molecular weight and proteins.

[0049] By use of the composition of the present invention, genes can beefficiently transferred into cells either in vivo or in vitro. In thecase of in vitro transfer, genes can be delivered into target cells bymeans of adding the composition of the present invention to a suspensioncontaining target cells, or by culturing target cells with mediumcontaining the composition of the present invention. In the case of invivo transfer, the composition of the present invention can beadministered into a host. Administration can be carried out eitherorally or parentally. Oral administration may be carried out by use ofconventional formulations therefor, such as tablets, powders, andgranules. Parental administration may be carried out by use ofconventional formulations therefor, such as injection, instillation,ointments, and suppositories. Among these, parenteral administration ispreferred; particularly, injection is most preferred; and for itsadministration, intravenous injection or local injection at target cellsites or organs is preferred.

EXAMPLES

[0050] Next, the present invention will be described in detail by way ofexamples, which should not be construed as limiting the invention.

Example 1 Production of Empty Liposomes Without a Gene

[0051] 1-1. Production of Empty-liposomes Dispersion

[0052] Predetermined amounts of a quaternary ammonium salt,phospholipid, and cholesterol were dissolved in chloroform, andsubsequently subjected to vacuum drying so as to obtain a lipid mixture.To the mixture, a predetermined amount of isotonic sucrose or lactosesolution was added, and subsequently, while being warmed up, the mixturewas subjected to emulsification by use of a homomixer. Thus, a crudeliposomal dispersion was obtained. Next, in order to adjust the particlesize of the liposomes, the liposome solution was subjected to extrusionprocedure under high pressure through a membrane filter having a poresize of 0.22 μm, to thereby obtain an empty-liposome dispersion.

[0053] 1-2. Production of Lyophilized Empty Liposomes

[0054] A predetermined amount of the empty-liposome dispersion preparedin 1-1 was aliquoted into vials, followed by lyophilization, to therebyobtain lyophilized liposomes.

Example 2 Production of Gene-containing Liposomes

[0055] 2-1. Production of a Gene-containing Liposomal Dispersion (type1)

[0056] An empty-liposome dispersion (2 μmol/ml of the total lipidconcentration) manufactured in 1-1 was diluted with serum-free medium(D-MEM) to a concentration of 100 nmol/ml (the quaternary ammonium saltconcentration). Next, 100 μl of the empty-liposome dispersion, whichcontained 10 nmol of the quaternary ammonium salt, and 14 μg DNA (eitherPGV-C (luciferase gene) or pCAG-lacZ (β-galactosidase gene)) were mixedwith 100 μl D-MEM and left for 15 min; and then 0.8 ml serum-free D-MEMsupplemented with 12.5% FBS (the final concentration of FBS was 10%) wasadded to the mixture so as to obtain a 1-ml sample.

[0057] 2-2. Production of a Gene-containing Liposomal Dispersion (type2)

[0058] The lyophilized dispersion of empty liposomes containing aconcentration equivalent to a 2 μmol/ml total lipid concentration, whichwas manufactured in 1-2, was rehydrated with distilled water to therebyreconstitute the original form (2 μmol/ml as a total lipidconcentration). Further, this solution was diluted with serum-freemedium (D-MEM) to 100 nmol/ml as a quaternary ammonium saltconcentration. Next, 100 μl of this liposomal dispersion containing a100 nmol equivalent quaternary ammonium salt and 1 μg DNA (PGV-C orpCAG-lacZ) was mixed with 100 μl serum-free medium (D-MEM), and left for15 min. Further, to the mixture, 0.8 ml D-MEM supplemented with 12.5%FBS (the final concentration of FBS was 10%) was added to obtain 1 ml ofsolution to thereby obtain a sample

[0059] 2-3. Production of a Gene-containing Liposome Dispersion (type 3)

[0060] Distilled water that containing DNA (PGV-C or pCAG-lacZ) wasadded for rehydration (1 μg DNA/10 nmol of the quaternary ammonium salt)to the lyophilized empty liposomes manufactured in 1-2 (2 μmol/mlequivalent as the total lipid concentration), and left for 15 min. Thenthe mixture was diluted to a final concentration of 1 μg/ml DNA withD-MEM supplemented with 10% FBS to thereby obtain a sample.

[0061] 2-4. Production of a Gene-containing Liposomal Dispersion (type4)

[0062] The empty liposomal dispersion manufactured in 1-1 (2 μmol/ml asthe total lipid concentration) was diluted to 400 nmol/ml as thequaternary ammonium salt with serum-free medium (D-MEM). Next, 500 μl ofthis liposomal dispersion, containing 200 nmol of the quaternaryammonium salt, and 500 μl of serum-free medium (D-MEM) containing 20 μgDNA (pCAG-lacZ) were mixed, and left for 5 min., to thereby obtain asample.

[0063] 2-5. Production of a Gene-containing Liposomal Dispersion (type5)

[0064] To the lyophilized empty liposomes equivalent to 2 μmol/ml of thetotal lipid and manufactured in 1-2, distilled water was added forrehydration to thereby reconstitute the original form (2 μmol/ml of thetotal lipid concentration), and the solution was further diluted withserum-free medium (D-MEM) so as to adjust the concentration to 400nmol/ml as the quaternary ammonium salt. Next, 500 μl of this liposomaldispersion, containing 200 nmol of the quaternary ammonium salt, and 500μl of serum-free medium (D-MEM) containing 20 μg DNA (pCAG-lacZ) weremixed and left for 5 min, to thereby obtain a sample.

[0065] 2-6. Production of a Gene-containing Liposomal Dispersion (type6)

[0066] To the lyophilized empty liposomes equivalent to 2 μmol/ml of thetotal lipid and manufactured in 1-2, distilled water containing DNA(pCAG-lacZ) was added (1 μg DNA/10 nmol of the quaternary ammonium salt)for rehydration, and left for 15 min. Further, this liposomal dispersionwas diluted to a final concentration of 20 μg/ml DNA with serum-freemedium (D-MEM), to thereby obtain a sample.

[0067] 2-7. Production of a Gene-containing Liposomal Dispersion (type7)

[0068] pCAG-lacZ DNA contained in the sample of the gene-containingliposomal dispersion (type 5) manufactured in 2-5 was replaced withpCAG-TK (a thymidine kinase gene). In all other respects, the type 7sample was manufactured in the same manner as was the type 5 sample.

Test Example 1 Measurement of Luciferase Activity

[0069] Respective types of tumor cells were plated onto 6-well plates ata concentration of 1×10⁵−8×10⁵ cells/well, and cultured for 24 h inmedium supplemented with 10% FBS, after which each well was washed oncewith serum-free medium. Then, 1 ml of the liposomal dispersioncontaining-PGV-C (see 2-1, 2-2, and 2-3; the final concentration was 1μg DNA/10 nmol of the quaternary ammonium salt/ml) was added to eachwell, and reacted at 37° C. for 5 h. Then, after each well was washedwith serum-free medium once, culture medium supplemented with 10% FBSwas added to each well; and, after cells were further cultured for 2days, luciferase assay was carried out.

[0070] Luciferase assay was performed as described below. Each well waswashed twice with phosphate-buffered saline (−) [PBS (−)]. Then, 150 μlof a cell-solubilizing solution (LCβ) was added to each well, and thewell plates were left at room temperature for 15 min. Then the cellswere scraped off from the plate substrate by use of a cell scraper. Eachlysate was subjected to centrifugation at 12,000 rpm for 2 min. Uponmixing 20 μl of the supernatant with 100 μl of a luminescent reagent,luminescence was measured by use of a lumi photometer (TD-4000,Laboscience). The amount of protein in each sample was estimated by useof BCA Protein Assay Reagent. Luciferase activity was expressed asemitted amount/mg protein. The results are shown in Tables 1 and 2.TABLE 1 In vitro luciferase activity obtained through use of differentliposomes (D-MEM medium supplemented with 10% FBS) Composition ofliposomal membrane Method for [Cationic lipid preparing a Luciferaseactivity appears first] liposomal (light units/mg protein sec) [Membranecomposition disper-sion Colo320 mEIIL HEC-1A HRA ES-2 props. in Exs. areon containing a (colon (uterus (uterus (ovary (ovary the mole basis.]Remarks gene cancer) cancer) cancer) cancer) cancer) Comp. CommercialCompositional lipid film⁷⁾ 489 838 3185 643 245 Ex. 1 Genetransfer prop.of the (Wako Pure Chemicals) membrane; 1) Comp. Commercial Compositionalcomplex⁸⁾ 176 3933 944 Ex. 2 LipofectACE prop. of the (LifeTechnologies) membrane; 2) Comp. Commercial Compositional Complex 429Ex. 3 LipofectAMlNE prop. of the (Life Technologies) membrane; 3) Comp.Commercial Compositional Complex 70 Ex. 4 LIPOFECTIN prop. of the (LifeTechnologies) membrane; 4) Comp. Commercial Compositional Complex 143Ex. 5 DMRIE-C prop. of the (Life Technologies) membrane; 5) Comp. LeafHuang^(a)) Compositional Complex 8 6 0 Ex. 6 prop. of the membrane; 6)Comp. SA^(b))/DOPE^(c))/DLPC^(d)) =2/4/4 type 3 8 194 0 Ex. 7 Comp. onlyDC-6-14^(e) Cationic type 1 4 0 Ex. 8 lipid alone Ex. 1 DC-6-12/DOPE =5/5 type 1 869 Ex. 2 DC-6-14/DOPE = 5/5 type 1 2671 4877 1237 Ex. 3DC-6-16/DOPE = 5/5 type 3 11823 1423 Ex. 4 DC-6-16/DOPE = 4/6 type 3 929Ex. 5 DC-6-12/DOPE/Chol^(f)) = 4/3/3 type 1 1244 323 Ex. 6DC-6-14/DOPE/Chol = 4/3/3 type 1 3096 103503 71498 1682 Ex. 7DC-6-14/DOPE/Chol = 4/3/3 type 2 71029 Ex. 8 DC-6-16/DOPE/DLPC = 4/2/4type 1 11930 Ex. 9 DC-6-14/DOPE/Chol = 2.9/4.2/2.9 type 1 466 6111 1094Ex. 10 DC-6-14/DOPE/Chol = 3.6/3.6/2.8 type 1 2455 10776 1070 Ex. 11DC-6-14/DOPE/Chol = 4.6/1.8/3.6 type 1 1607 3161 421 Ex. 12DC-6-14/DOPE/Chol = 5/2/3 type 1 1111 3532 261 Ex. 13 DC-6-16/DQPE/DLPC= 3/4/3 type 2 469 629 Ex. 14 DC-6-16/DOPE/DLPC = 4/4/2 type 2 423 1923Ex. 15 DC-6-18:1/DOPE/DLPC = 2/4/4 type 2 785 Ex. 16 DC-6-18:1/DOPE/Chol= 2/4/4 type 2 2951

[0071] a) See Biochem. Biophys. Res. Comm., vol. 179, No. 1, 280-285(1991)

[0072] b) SA: Stearylamine (typical cationic lipid)

[0073] c) DOPE: Dioleylphosphatidylethanolamine

[0074] d) DLPC: Dilauroylphosphatidylcholine

[0075] e) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chlorideDC-6-14:O,O,′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride DC-6-16:O,O,′-N-dihexadecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride DC-6-18:1:O,O′-N-dioctadecenoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

[0076] f) Chol: cholesterol

[0077] 1)N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4 (moleratio)

[0078] 2) Dimethyloctadecylammonium bromide/DOPE=2.9/7.1 (weight ratio)

[0079] 3)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1 (weight ratio)

[0080] 4) N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride/DOPE=5/5 (weight ratio)

[0081] 5) 1,2-Dimyristyloxypropyl-3-dimethylhydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

[0082] 6)3-β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol/DOPE=6/4 (moleratio)

[0083] 7) The commercial product used was a “lipid film.” Agene-containing solution (aqueous solvent) was added to the “lipid film”so that 1 μg DNA was contained per 10 nmol of cationic lipid containedin the component, followed by mixing in a vortex mixer, to therebyobtain a gene-containing aqueous liposomal dispersion.

[0084] 8) The commercial product and the method described in theliterature: To an aqueous dispersion of empty liposomea (or a lipidmembrane structure), a gene-containing solution (aqueous solvent) wasadded so that 1 μg DNA was contained per 10 nmol of cationic lipidcontained in the component, to thereby obtain a dispersion ofgene-liposome complex. TABLE 2 In vitro luciferase activity obtainedthrough use of different liposomes (D-MEM medium supplemented with 10%FBS) Composition of liposomal membrane Method for [Cationic lipidpreparing a Luciferase activity appears first] liposomal (light units/mgprotein sec) [Membrane composition dispersion Colo320 HEC-1A HRA KFprops. in Exs. are on containing (colon (uterus (ovary (ovary the molebasis.] Remarks a gene cancer) cancer) cancer) cancer) Comp. CommercialCompositional lipid film⁷ 489 3185 643 23 Ex. 1 Genetransfer prop. ofthe (Wako Pure Chemicals) membrane;1) Comp. Commercial LipofectACECompositional complex⁸ 176 3933 944 Ex. 2 (Life Technologies) prop. ofthe membrane; 2) Comp. Commercial Compositional Ex. 3 LipofectAMINEprop. of the Complex 429 (Life Technologies) membrane; 3) Comp.Commercial LIPOFECTIN Compositional Ex. 4 (Life Technologies) prop. ofthe Complex 70 membrane; 4) Comp. Commercial DMRIE-C Compositional Ex. 5(Life Technologies) prop. of the Complex 143 membrane; 5) Comp. LeafHuang^(a)) Compositional Complex 8 6 0 Ex. 6 prop. of the membrane; 6)Comp. SA^(b))/DOPE^(c))/DLPC^(d)) = 2/4/4 type 3 8 194 0 Ex. 7 Ex. 17TC-1-12^(g))/DOPE/DLPC = 5/5 type 3 2887 342 Ex. 18 TC-1-12/DOPE/DLPC =2/4/4 type 1 8325 6245 Ex. 19 TC-1-12/DOPE/DLPC = 3/4/3 type 2 5429 Ex.20 TC-1-12/DOPE/DLPC = 3/4/3 type 3 3459 1204 40 Ex. 21TC-1-12/DOPE/lysoPC^(h))= 2/4/4 type 3 1598 Ex. 22 TC-1-12/DOPE/Chol =5/2.5/2.5 type 3 2176 Ex. 23 TC-1-12/DOPE/Chol/DLPC = 2/4/2/2 type 33909 89 Ex. 24 TC-1-12/DOPE = 4/6 type 2 1378 Ex. 25 TC-1-12/DOPE/DLPC =4/4/2 type 2 3355 446 648 Ex. 26 TC-1-12/DOPE/DLPC = 5/4/1 type 2 2897414 488 Ex. 27 TC-1-12/DOPE/Lyso-LPC^(i)) = 2/4/4 type 2 4499 Ex. 28TC-1-12/DOPE/LYSO-MPC^(j)) = 2/4/4 type 2 3258 Ex. 29 TC-1-12/DOPE/Chol= 2.5/2.5/5 type 2 1680 223 Ex. 30 TC-1-12/DOPE/Chol = 2.5/5/2.5 type 21606 287 Ex. 31 TC-1-12/DOPE/Chol = 5/4.5/0.5 type 2 1221

[0085] a)-f) and 1)-8): See Table 1

[0086] g) TC-1-12: O,O′,O″-tridodecanoyl-N-({overscore(ω)}-trimethylammoniodecanoyl)-tris(hydroxymethyl)-aminomethane bromide

[0087] h) LysoPC: Lisophosphatidylcholine

[0088] i) Lyso-LPC: Lisolauroylphosphatidylcholine

[0089] j) Lyso-MPC: Lisomyristoylphosphatidylcholine

[0090] As is apparent from Tables 1 and 2, the gene transfer compositionaccording to the present invention exhibited enhanced gene transferability as compared with commercial gene transfer reagents.

Test Example 2 X-gal Staining

[0091] Each type of tumor cells were plated in amounts of 1×10⁵−8×10⁵ ina six-well plate, followed by culturing for 24 hours in a medium addedwith FBS (10%) and washing once with serum-free medium. Subsequently, 1ml of a gene-containing liposomal dispersion (gene: pCAG-lacZ) preparedin Example 2 (2-1, 2-2, or 2-3; final DNA concentration=1 μg/10 nmolquaternary ammonium salt/ml) was added to each well, and reaction wasallowed to proceed for five hours. When five hours have elapsed, thewells were washed once with serum-free medium. FBS (10%)-supplementedmedium was added and subjected to incubation for two days, and thenX-gal staining.

[0092] X-gal staining was performed as follows. Briefly, the sample waswashed once with PBS(−), then fixed for 3-4 minutes by use of PBS(−)containing 1% formaldehyde, 0.2% glutaraldehyde, and 0.02% NP40,followed by washing PBS(−) three times, each for 10 minutes. Ultimately,staining was performed for 5-8 hours at 37° C. by use of a mixturesolution containing 5 mM K₄[Fe(CN)₆], 5 mM K₃[Fe(CN)₆], 0.01% sodiumdeoxycholic acid, 0.02% NP40, 2 mM MgCl₂, and 0.1% X-gal. Under amicroscope, cells were counted at least 1,000 in number, to therebyobtain a Lac Z-positive number. The results are shown in Tables 3through 5. TABLE 3 Percentage in vitro LacZ positive cells obtainedthrough use of different liposomes (in medium supplemented with 10% FBS)Composition of Percentage LacZ positive cells liposomal membrane Methodfor (count of positive cells per 100 cells) [CL appears first] preparinga HEC-1A mEIIL HRA ES-2 SW626 KF KOC-3S [MCP in Exs. are lipsomaldispersion (uterus (uterus (ovary (ovary (ovary (ovary (ovary on themole basis.] Remarks containing a gene cancer) cancer) cancer) cancer)cancer) cancer) cancer) Comp. Commercial CP of lipid film⁴ 2.6 7.6 10.18.6 9.2 3.3 6.0 Ex. 1 Genetransfer membrane; 1) Comp. Commercial CP ofcomplex⁵ 0.6 4.7 3.3 0 2.4 Ex. 3 LipofectAMINE membrane; 2) Comp.Commercial DMRIE-C CP of Complex 4.0 1.3 10.1 0.6 0.5 Ex. 5 membrane; 3)Ex. 5 DC-6-12^(a))/DOPE^(b))/ type 1 21.2 10.6 38.9 24.7 14.8 14.5 4.8Chol^(c)) = 4/3/3 Ex. 6 DC-6-14/DOPE/Chol = 4/3/3 type 1 42.1 23.7 16.05.9 Ex. 32 DC-6-12/DOPE/Chol = 4/3/3 type 3 8.7 8.4 9.3 13.7 Ex. 33DC-6-14/DOPE/Chol = type 1 14.9 21.4 7.4 4.8 1.8/5.4/2.8

[0093] TABLE 4 Percentage in vitro LacZ positive cells obtained throughuse of different liposomes (in medium supplemented with 10% FBS)Composition of Percentage LacZ positive cells liposomal membrane Methodfor (count of positive cells per 100 cells) [CL appears first] preparinga Nakajima Nakajima OVHS-1 SKOV-3 KK OVCAR 3 HNOA [MCP in Exs. are onliposomal dispersion (ovary S2 (ovary (ovary (ovary (ovary (ovary (ovarythe mole basis.] Remarks containing a gene cancer) cancer) cancer)cancer) cancer) cancer) cancer) Comp. Commercial CP of lipid film 6.05.4 0.9 6.6 31.3 8.5 1.4 Ex. 1 Genetransfer membrane; 1) Comp.Commercial CP of Complex 0.1 0.6 1.0 0.2 0 0.4 0 Ex. 3 LipofectAMINEmembrane; 2) Comp. Commercial CP of Complex 0.1 1.0 0 0.2 0 4.5 0 Ex. 5DMRIE-C membrane; 3) Ex. 5 DC-6-12^(a))/DOPE^(b))/ type 1 7.4 8.6 8.3Chol^(c)) = 4/3/3 Ex. 6 DC-6-14/DOPE/ type 1 6.5 11.5 12.3 Chol = 4/3/3Ex. 32 DC-6-12/DOPE/ type 3 4.6 Chol = 4/3/3 Ex. 33 DC-6-14/DOPE/ type 14.4 9.1 22.2 7.5 Chol = 1.8/5.4/2.8

[0094] TABLE 5 Percentage in vitro LacZ positive cells obtained throughuse of different liposomes (in medium supplemented with 10% FBS)Composition of Percentage LacZ positive cells liposomal membrane Methodfor (count of positive cells per [CL appears first] preparing a 100cells) [MCP in Exs. are liposomal dispersion HEC-1A COS-1 on the molebasis.] Remarks containing a gene (uterus cancer) (fibroblast cells)Comp. Commercial Genetransfer CP of lipid film 2.6 Ex. 1 (Wako PureChemicals) membrane; 1) Comp. Commercial LipofectAMlNE CP of Complex 0Ex. 3 (Life Technologies) membrane; 2) Ex. 20TC-1-12^(a))/DOPE^(b))/DLPC^(d)) = 3/4/3 type 3 11.5 Ex. 23TC-1-12/DOPE/Chol^(c))/DLPC = 2/4/2/2 type 3 18.0 Ex. 34TC-1-12/DOPE/DLPC = 2/4/4 type 3 14.1 Ex. 35 TC-1-12/DOPE/Chol = 3/4/3type 1 3.7 Ex. 36 TC-1-12/DOPE/Chol = 3/4/3 type 3 11.6 Ex. 37TC-1-12/DOPE/Chol/DLPC = 2/4/3/1 type 3 15.3

[0095] a) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chlorideDC-6-14:O,O,′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride DC-6-16: O,O,′-N-dihexadecanoyl-N-({overscore(ω)}-trimethylammonioacetyl)diethanolamine chloride TC-1-12:O,O′,O″-tridodecanoyl-N-(αtrimethylammoniodecanoyl) -tris(hydroxymethyl) -aminomethane bromide

[0096] b) DOPE: Dioleylphosphatidylethanolamine

[0097] c) Chol: Cholesterol

[0098] d) DLPC: Dilauroylphosphatidylcholine

[0099] 1)N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4 (moleratio)

[0100] 2)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1 (weight ratio)

[0101] 3) 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

[0102] 4) The commercial product used was a “lipid film.” Agene-containing solution (aqueous solvent) was added to the “lipid film”so that 1 μg DNA was contained per 10 nmol of cationic lipid containedin the component, followed by mixing in a vortex mixer, to therebyobtain a gene-containing aqueous liposomal dispersion.

[0103] 5) The commercial product and the method described in theliterature: To an aqueous dispersion of empty liposomes (or a lipidmembrane structure), a gene-containing solution (aqueous solvent) wasadded so that 1 μg DNA was contained per 10 nmol of cationic lipidcontained in the component, to thereby obtain a dispersion ofgene-liposome complex.

[0104] As is apparent from Tables 3 to 5, the gene transfer compositionaccording to the present invention exhibited enhanced gene transferability as compared with commercial gene transfer reagents.

Test Example 3 X-gal Staining (2)

[0105] Each type of tumor cells were intraperitoneally inoculated toeach nude mouse in amounts of 5×10⁶ (mEIIL, ES-2) -6×10⁷ (HRA). Afterone day (in the case of HRA), about 10 days (in the case of ES-2), orabout 3 weeks (in the case of mEIIL), 1 ml of the gene-containingliposomal dispersion (gene: pCAG-lacZ) described above (2-4, 2-5, or2-6; final DNA concentration=20 μg/200 nmol quaternary ammonium salt/ml)was intraperitoneally administered to the mouse. On the following day(in the case of mEIIL and HRA) or two days later (in the case of ES-2),tumor cells were collected, and 3×10⁵⁻5×10⁵ cells were plated in thewells of a 6-well plate. The cells were incubated for 24 hours by use ofa medium supplemented with 10% FBS, followed by X-gal staining. X-galstaining was performed in a manner similar to that in Test Example 2.The results are shown in Table 6. TABLE 6 Percentage in vivo LacZpositive cells obtained through use of different liposomes Compositionof Percentage Lac Z positive cells liposomal membrane Method for (countof cells per 100 cells) [CL appears first] preparing a HRA [MCP in Exs.are liposomal dispersion HRA mEIIL ES-2 on the mole basis.] Remarkscontaining a gene (ovary cancer) (uterus cancer) (ovary cancer) Comp.Commercial Genetransfer CP of Lipid film⁶⁾ 0.95 0.25 0.25 Ex. 1membrane; 1) Comp. Commercial LipofectACE CP of complex⁷⁾ 0.62 Ex. 2membrane; 2) Comp. Commercial LipofectAMINE CP of Complex 0.23 Ex. 3membrane; 3) Comp. Commercial LIPOFECTIN CP of Complex 0.38 Ex. 4membrane; 4) Comp. Commercial DMRIE-C CP of Complex 1.52 Ex. 5 membrane;5) Ex. 38 DC-6-12^(a))/DOPE^(b)) = 5/5 type 4 5.50 Ex. 39 DC-6-14/DOPE =5/5 type 5 1.08 Ex. 40 DC-6-14/DOPE = 5/5 type 6 1.04 Ex. 41DC-6-12/DOPE/Chol^(c)) = 4/3/3 type 4 4.71 1.29 2.43 Ex. 42DC-6-14/DOPE/Chol = 4/3/3 type 4 1.32 Ex. 43 DC-6-14/DOPE/Chol = 4/3/3type 6 0.96 Ex. 44 DC-6-14/DOPE/Chol = 1.8/5.4/2.8 type 4 4.32 Ex. 45DC-6-14/DOPE = 4/6 type 4 6.30

[0106] a) DC-6-12:O,O,′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chlorideDC-6-14:O,O′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

[0107] b) DOPE: Dioleylphosphatidylethanolamine

[0108] c) Chol: cholesterol

[0109] 1)N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4 (moleratio)

[0110] 2) Dimethyldioctadecylammonium bromide/DOPE=2.9/7.1 (weightratio)

[0111] 3)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1(weight ratio)

[0112] 4) N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride/DOPE=5/5 (weight ratio)

[0113] 5) 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

[0114] 6) The commercial product used was a “lipid film.” Agene-containing solution (aqueous solvent) was added to the “lipid film”so that 1 μg DNA was contained per 10 nmol of cationic lipid containedin the component, followed by mixing in a vortex mixer, to therebyobtain a gene-containing aqueous liposomal dispersion.

[0115] 7) The commercial product and the method described in theliterature: To an aqueous dispersion of empty liposomes (or a lipidmembrane structure), a gene-containing solution (aqueous solvent) wasadded so that 1 μg DNA was contained per 10 nmol of cationic lipidcontained in the component, to thereby obtain a dispersion ofgene-liposome complex.

[0116] As is apparent from Table 6, the gene transfer compositionaccording to the present invention exhibited enhanced gene transferability as compared with commercial gene transfer reagents.

Test Example 4 Life-prolonging Effect in Tumor-bearing Mice

[0117] Each type of tumor cells were intraperitoneally inoculated tonude mice in amounts of 3×10⁵ (HRA), 1×10⁶ (mES-2), or 5×10⁶ (day 0).From day 7 (in cases of HRA and ES-2) or from day 10 (in the case ofmEIIL), 1 ml of the gene-containing liposomal dispersion (gene: pCAG-TK)described above (2-7; final DNA concentration=20 μg/200 nmol quaternaryammonium salt/ml) was intraperitoneally administered to each mouse. Foreach of 13 consecutive days starting from day 9, i.e., until day 21 (incases of HRA and ES-2), or for each of 13 consecutive days starting fromday 12, i.e., until day 24 (in the case of mEIIL), acyclovir wasintraperitoneally administered to each mouse twice a day at a dose of 35mg/kg. To mice of a control group, pCAG-lacZ gene was administered inplace of pCAG-TK. The significance test employed was the Cox-Mantelmethod. The results are shown in Table 7. TABLE 7 Life-prolonging effectof liposomes in a peritoneum-implanted model Composition of Survivedmice liposomal membrane Count of Number Days of Days of at the end ofSignificance (mole ratio) Gene Oncocyte implanted cells of miceobservation 50% Survival observation test Ex. 46 DC-6-14^(a))/DOPE^(b))= 5/2 pCAG-TK HRA 3 × 10⁵ 12 70 40 5 p < 0.05 (C.G.) DC-6-14/DOPE = 5/2pCAG-lacZ HRA 3 × 10⁵ 12 70 34 1 Ex. 47 DC-6-14/DOPE = 5/2 pCAG-TK mEIIL5 × 10⁶ 8 85 85 or 6 p < 0.05 more (C.G.) DC-6-14/DOPE = 5/2 pCAG-lacZmEIIL 5 × 10⁶ 8 85 67 1 Ex. 48 DC-6-14/DOPE/Chol^(c)) = pCAG-TK mEIIL 5× 10⁶ 8 85 85 or 6 p < 0.05 1.8/5.4/2.8 more (C.G.) DC-6-14/DOPE/Chol =pCAG-lacZ mEIIL 5 × 10⁶ 8 85 60 2 p < 0.05 1.8/5.4/2.8 Ex. 49DC-6-12/DOPE/Chol = pCAG-TK ES-2 1 × 10⁶ 12 77 50 3 4/3/3 (C.G.)DC-6-12/DOPE/Chol = pCAG-lacZ ES-2 1 × 10⁶ 12 77 37 0 4/3/3

[0118] a) DC-6-12:O,O,′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chlorideDC-6-14:O,O,′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine-chloride

[0119] b) DOPE: Dioleylphosphatidylethanolamine

[0120] c) Chol: cholesterol

[0121] As is apparent from Table 7, the gene transfer compositionaccording to the present invention exhibited excellent life-prolongingeffect.

Industrial Applicability

[0122] The composition of the present invention enables effectivedelivery and expression of a gene which previously could not beeffectively expressed in a cell due to the low ratio at which the geneis introduced into cells. Therefore, the composition is advantageouslyused as a gene transfer reagent or a pharmaceutical.

1. A composition for gene transfer into cells, which compositioncomprises a quaternary ammonium salt represented by the followingformula (1):

(wherein each of R¹, R², R³, R⁴, and R₅, which are identical to ordifferent from one another, represents a C9-C17 aliphatic group); X¹represents a halogen atom; and n is an integer from 1 to 10 inclusive.2. A composition according to claim 1, wherein in

(wherein R¹ and R² have the same meanings as defined above) and n is 1.3. A composition according to claim 1, wherein in

(wherein R³, R⁴, and R⁵ have the same meanings) and n is
 10. 4. Acomposition according to any one of claims 1 through 3, wherein informula (1), R¹, R², R³, R⁴, and R⁵ are identical to or different fromone another, and each represents a C9-C17 linear alkyl group.
 5. Acomposition according to any one of claims 1 through 3, wherein informula (1), R¹, R², R³, R⁴, and R⁵ are identical to or different fromone another, and each represents a C11-C15 linear alkyl group.
 6. Acomposition according to any one of claims 1 through 5, which furthercomprises a phospholipid and/or cholesterol.
 7. A composition accordingto claim 6, wherein the phospholipid is one or more members selectedfrom the group consisting of phosphatidylethanolamine,phosphatidylcholine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, cardiolipin, sphingomyelin, plasmalogen, andphosphatidic acid.
 8. A composition according to claim 6, wherein thephospholipid is one or more members selected from the group consistingof phosphatidylethanolamine and phosphatidylcholine.
 9. A compositionaccording to any one of claims 6 through 8, which forms liposomes.
 10. Acomposition according to any one of claims 1 through 9, which furthercomprises a gene.
 11. A method for gene transfer into cells, whichcomprises applying the composition as described in claim 10 to cells invitro or in vivo.